System and method for in vitro blood vessel modeling

ABSTRACT

The present invention provides an in vitro blood vessel model for investigation of drug induced vascular injury and other vascular pathologies. The in vitro blood vessel model provides two channels separated by a porous membrane that is coated on one side by an endothelial cell layer and is coated on the other side by a smooth muscle cell layer, wherein said model is susceptible to the extravasation of red blood cells across said porous membrane due to drug induced vascular injury.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application61/065,356, filed Feb. 11, 2008, and U.S. Provisional Application61/103,117, filed Oct. 6, 2008, both of which are incorporated herein intheir entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to a system and method for creating an invitro blood vessel model to investigate drug induced vascular injury andother vascular pathology.

BACKGROUND OF THE INVENTION

Blood vessels are a tissue central to many disease states. One suchdisease state is drug induced vascular injury (DIVI), a pathologicalinsult to blood vessels that occurs after the administration of a drug.DIVI causes smooth muscle cell (SMC) death and hemorrhage and ischaracterized by the extravasation of red blood cells (RBCs) from thevascular lumen into surrounding SMC layers. This phenomenon occurswithout overt damage to the endothelial cells (ECs). The cause of DIVIis poorly understood but its occurrence often halts in vivo testing ofcandidate drugs.

The predilection for DIVI is unclear. The central and only specificevent in DIVI is the extravasation of RBCs into the media of theeffected blood vessels. Currently, the molecular mechanisms of DIVI areunclear and no specific biomarkers are known that allow DIVI to bedistinguished from other forms of vascular injury. It is contemplatedthat improved understanding of the mechanisms of DIVI will improve thescreening of drugs under development and better correlate thesignificance of in vivo animal testing and human susceptibility to DIVI.

It would therefore be desirable to develop an in vitro model of a smalldiameter blood vessel to allow the study of a variety of vasculardiseases and physiological mechanisms. In particular, the model would beused to investigate the mechanisms and biomarkers of DIVI in animals andhumans. It would provide a platform for the screening of drugs underdevelopment and allow disease processes effecting small to medium sizedvessels to be investigated.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides an in vitro bloodvessel model comprising two channels separated by a porous membrane thatis covered on one side by endothelial cells and is covered on the otherside by smooth muscle cells, wherein said model is susceptible to theextravasation of red blood cells across said porous membrane due to druginduced vascular injury.

The porous membrane may be designed to mimic the internal elastic laminaof blood vessels, preferably having a porosity of approximately 11%,pore sizes ranging from 2.3-13 μm, and an average pore size of 6.4 μm.It is contemplated that this porous membrane is a track-etchedpolycarbonate membrane that may be coated with at least one of elastin,fibrinogen, and collagen, or combinations thereof.

The in vitro blood vessel model includes a luminal channel designed tomimic the lumen of a small blood vessel and a smooth muscle channeldesigned to mimic the medial layer of a blood vessel. It is alsocontemplated that, in the preferred embodiment, the smooth musclechannel is an open smooth muscle channel that may be capped by a lid andmay include beveled walls adjacent to the porous membrane.

In a certain embodiment of the present invention, the in vitro bloodvessel model includes smooth muscle and luminal channels having offsetinflow and outflow ponds that reduce non-physiological shear stress onthe porous membrane.

In another aspect of the present invention, a method of assaying anagent for its ability to cause drug induced vascular injury is provided.Such a method comprises, providing an in vitro blood vessel modelcomprising two channels separated by a porous membrane that is coated onone side by an endothelial cell layer and is coated on the other side bya smooth muscle cell layer, wherein said model is susceptible to theextravasation of red blood cells through said porous membrane due todrug induced vascular injury, passing the agent through the channeladjacent the endothelial cells of the in vitro model, and analyzing thechannel adjacent the smooth muscle cells for the extravasation of redbloods cells, which indicates that the agent causes drug inducedvascular injury.

These and other features, objects and advantages of the presentinvention will become better understood from the description thatfollows. The description of preferred embodiments is not intended tolimit the invention to cover all modifications, equivalents andalternatives. Reference should therefore be made to the claims recitedherein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a perspective view of a microfluidic device according toone embodiment of the present invention, including a closed smoothmuscle channel;

FIG. 2 shows a perspective view of a microfluidic device according toone embodiment of the present invention, including an open smooth musclechannel with beveled walls;

FIGS. 3 a and 3 b respectively show an open smooth muscle channel withand without a lid in accordance with one embodiment of the presentinvention;

FIG. 4 shows a bifurcated luminal channel in accordance with oneembodiment of the present invention;

FIG. 5 shows one embodiment of the present invention, including offsetponds;

FIG. 6 shows a process that may be used to manufacture the presentinvention.

FIG. 7 shows an immunohistochemistry stain of F-actin on smooth musclecells grown in an in vitro blood vessel model in accordance with thepresent invention; and

FIG. 8 shows an immunohistochemistry stain of CD31 and DAPI onendothelial cells grown in an in vitro blood vessel model in accordancewith the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides an in vitro model of a blood vessel,through which blood or culture media flows at physiological rates, thatallows the investigation of a variety of vascular diseases andphysiological mechanisms.

Referring to FIGS. 1-2, the present invention includes a microfluidicdevice 10 having a luminal channel 12 separated from a smooth musclechannel 14 by a porous membrane 16.

The luminal channel 12 models the lumen of a blood vessel and may alsobe referred to as the vascular channel or vascular lumen. The smoothmuscle channel 14 is designed to allow SMC growth and models the mediallayer of the blood vessel. It is contemplated that SMC growth may bebetter facilitated by employing an open smooth muscle channel 22, whichprovides access to increased volumes of growth media, and beveled sides24, which allow cells to more readily come into contact with themembrane. This arrangement provides more uniform seeding of the deviceand minimizes the number of cells which attempt to attach to thenon-membrane portion of the smooth muscle channel 14. Referring to FIG.3, the open smooth muscle channel 22 may be capped with a lid 50 tocreate a large, media volume to promote SMC growth while improvingusability and sterile culture conditions. The lid 50, which is shownremoved in FIG. 3 a and applied in FIG. 3 b, can be applied before orafter SMC seeding.

Referring to FIGS. 1 and 4, it is contemplated that the vascular andsmooth muscle channels, 12 and 14 respectively, have a round orhydraulic diameter of approximately 200 μm, as DIVI typically occurs inmesenteric vessels of this size. Other diameters are possible and thepresent invention may also include branching channels with single ormultiple bifurcations. For example, the luminal channel 12 may include abifurcation 44 providing a second access 46 that allows luminal channelaccess without disturbing any tubing connections used to establish tocreate flow. In such a configuration, media flows through the central,linear portion of the luminal channel 12 and mimics physiological shearstress within the blood vessel lumen. Cells for may then be seeded intothe blood vessel lumen using the second access 46 and a fitting adaptedfor seeding cells without disturbing the central channel's flowconnections. It is contemplated that the second access 46 can also beused to inject drugs or other reagents.

Referring again to FIG. 1, the luminal and smooth muscle channels areseparated by a porous membrane 16 that approximates the internal elasticlamina of blood vessels. The internal elastic lamina is an extracellularmatrix layer disposed between the endothelial cells of the intima andthe smooth muscle cells of the media. It is contemplated that the porousmembrane 16 is a track-etched polycarbonate membrane coated with atleast one of elastin, fibronectin, fibrin, laminin, hyaluronic acid, andcollagen. A preferred embodiment may have a porosity of approximately11%, pore sizes ranging from 2.3-13 μm, and an average pore size ofapproximately 6.4 μm. However, a range of porosities betweenapproximately 5 and 50% and a range of pore sizes between 0.1 and 30 μmare possible. Moreover, the variance in pore size can also range and, insome cases, may be reduced to negligible levels. SMCs are seeded uponthe side of the porous membrane 16 that faces the smooth muscle channel14 to create a SMC membrane layer. Following seeding of SMCs, ECs areseeded upon the luminal side of the porous membrane 16 and are growninto an EC membrane layer. It is contemplated that the portion of theporous membrane adjacent to the ECs may be coated with differentcombinations or concentrations of extracellular matrix proteins than theporous portion of the membrane adjacent to the endothelial cells.

Blood flow through the device 10 at physiological flow rates generatesphysiological shear stress and causes RBCs to extravasate through theporous membrane 16 if the membrane does not include endothelial orsmooth muscle cell layers. However, when ECs and SMCs are grown onopposing sides of the membrane, culture media including RBC's or 6 μmmicrospheres (that is, RBC analogs) flow through the luminal channel 12,but do not extravasate through the porous membrane 16 into the smoothmuscle channel 14. Any combination of drugs and hemodynamic models canbe applied to this model to investigate the mechanisms and potentialbiomarkers of drug induced vascular injury.

In another embodiment, the present invention provides an in vitro bloodvessel model having 0.5-10 μm thick porous membrane including one ormore proteins such as collagen, elastin, fibrin, fibronectin, laminin,hylauronic acid. This membrane, is initially less porous than theinternal elastic lamina of a blood vessel, but, over time, is brokendown or remodeled by the endothelial and/or smooth muscle cells toproduce a membrane containing physiological proteins that allows theextravasation of RBCs or RBC analogs under appropriate conditions. Thisadvantageously allows the continued adhesion of ECs to the membraneunder high shear stress conditions. The membrane may further include anelectrospun material, for example, polycaprolactone, coated withextracellular matrix proteins such as collagen. Alternately, the presentinvention can include a porous collagen membrane that allows theextravasation of RBCs. This porous collagen membrane may be crosslinkedand may resist significantly remodeling by the cells over the course ofthe culture or experiment.

Additional cells can be cultured within or on the SMCs to further studyvascular physiology or pathological states that occur in blood vessels,such as atherosclerosis and inflammation. For example, fibroblasts maybe seeded above SMCs to replicate an adventitial-like layer to the bloodmodel. The fibroblasts can be seeded into the SMC channel shortly afterthe SMCs have been seeded or after several days of SMC culture. Inaddition, adipocytes can be seeded above the SMCs or following seedingwith fibroblasts. The adipocytes, with or without additionalfibroblasts, allow the establishment of perivascular fat tissue similarto an in vivo condition.

Referring now to FIG. 5, one embodiment of the microfluidic device 10has inlet and outlet areas for the microfluidic channels that are knownas ponds. Traditional microfluidic devices have overlapping ponds, butblood flow into overlapping ponds generates non-physiological shearstress on the porous membrane and a velocity vector directing fluid flowthrough the porous membrane. This arrangement can cause abnormal RBCextravasation and is not suitable for DIVI or vascular pathologytesting, as it may obscure test results within the model. The presentinvention instead employs a vascular channel pond 34 offset from asmooth muscle channel pond 36. As a result, fluid force no longer causesRBC extravasation into the smooth muscle channel 14 at the pond, as theporous membrane 16 over the vascular channel pond 34 abuts a solidmaterial.

Referring again to FIG. 3, one embodiment of the present inventionincludes tubes 52 that provide fluid access to the endothelial andsmooth muscle channels. The tubes in communication with the smoothmuscle channel 54 allow media change and at least one of the tubes maybe connected to a sterile filter (for example, a syringe filter) andelevated to create an atmospheric pressure vent.

Referring to FIG. 6, it is contemplated that the microfluidic device ismade from polydimethylsiloxane (PDMS) using soft photolithographytechniques. This process includes creating two layers with half-depthchannels 40 using a positive mold 42. Round or rectangular channels aremade using a rectangular or half-round shaped mold, respectively.

In another embodiment, the microfluidic device may be placed in a singlepass or recirculating system where culture media is passed through thedevice. The system determines and adjusts flow, overall pressure, andpressure waveform at the inlet and outlet of the device to produce adesired set of conditions, for example, physiologic conditions orpathological conditions observed in DIVI. These hemodynamic conditions,in addition to the presence or absence of a drug, may lead to animproved understanding of the mechanisms of DIVI.

Referring to FIGS. 7 and 8, an in vitro blood vessel model in accordancewith the present invention was used to study extravasation. This modelincluded bifurcated inlets and a track-etched polycarbonate membranehaving 10 μm pores. To study extravasation, both sides of the membranewere coated with type I rat tail collagen and primary rat aortic SMCswere then seeded within the open smooth muscle channel, attaching to theexposed membrane, but not attaching the PDMS regions. Following SMCseeding, a lid was applied to the top of the device to seal the SMCchannel and a syringe filter was attached to the outlet tubing to ventthe SMC channel to the atmosphere. As shown in FIG. 7, the SMCs werethen stained with phalloidin for F-actin to demonstrate initial SMCcoverage. After four days of SMC culture, dTomata-labeled primary rataortic ECs were seeded into a 200 μm luminal channel and, four hoursafter EC seeding, flow with the 200 μm luminal channel was initiatedusing a syringe pump. Over the next seven days flow was increased toarterial flow rates, that is, approximately 15 dynes/cm² or 7200 μm/hr.Confluency of the cell layers was then demonstrated by flowing 6 μmdiameter fluorescent microspheres through the device and evaluating beadextravasation into the smooth muscle layer or channel. The ECs and SMCswere the fixed and stained for CD31 and F-actin respectively.

The dTomato-labeled ECs indicated that the ECs formed a monolayer on thepolycarbonate membrane within the luminal channel channel. This wasfurther demonstrated by subsequent CD31 and DAPI(4′,6-diamidino-2-phenylindole) staining, as shown in FIG. 8. This testdemonstrated cell layer confluence and the physiological robustness ofthe model, as no extravasation of the 6 μm microspheres occurred after 2minutes of flow.

DIVI is only one example of a pathological or physiological processwhich can be investigated with the model. For example, atherosclerosiscould be modeled in part using ECs, SMCs, and perhaps other cell typessuch as macrophages or foam cells. Virtually any other disease processwhich effects small to medium sized vessels could be investigated usingthis model.

The present invention has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of theinvention.

1. An in vitro blood vessel model comprising two channels separated by a porous membrane coated on one side by an endothelial cell layer and coated on the other side by a smooth muscle cell layer, wherein the model is susceptible to the extravasation of red blood cells across the porous membrane due to drug induced vascular injury.
 2. The in vitro blood vessel model of claim 1 wherein the porous membrane has pore sizes ranging from approximately 0.1 to 30 μm and a porosity between approximately 5 and 50 percent.
 3. The in vitro blood vessel model of claim 2 wherein said porous membrane has an 11 percent porosity, pore sizes ranging from 2.3 to 13 μm, and an average pore size of 6.4 μm.
 4. The in vitro blood vessel model of claim 3 wherein said porous membrane is a track-etched polycarbonate membrane.
 5. The in vitro blood vessel model of claim 4 wherein said porous membrane is coated with at least one of elastin, fibrin, fibronectin, laminin, hyaluronic acid, and collagen.
 6. The in vitro blood vessel model of claim 1 wherein said two channels are a luminal channel mimicking the lumen of a small blood vessel and a smooth muscle channel mimicking the medial layer of a blood vessel.
 7. The in vitro blood vessel model of claim 6 wherein at least one of the luminal channel and the smooth muscle channel includes at least one bifurcation.
 8. The in vitro blood vessel model of claim 7 wherein said smooth muscle channel is an open smooth muscle channel including beveled walls adjacent to the porous membrane.
 9. The in vitro blood vessel model of claim 8 wherein said open smooth muscle channel may be capped with a lid to seal the smooth muscle channel from an external atmosphere.
 10. The in vitro blood vessel model of claim 1 wherein said two channels include offset inflow and outflow ponds that reduce non-physiological shear stress on the porous membrane.
 11. The in vitro blood vessel model of claim 1 wherein fibroblasts are seeded on the smooth muscle cell layer to replicate an adventitial layer.
 12. The in vitro blood vessel model of claim 1 wherein adipocytes are seeded on the smooth muscle cell layer to create perivascular fat tissue.
 13. The in vitro blood vessel model of claim 6 wherein the porous membrane is initially less porous than an internal elastic lamina of a blood vessel, but is remodeled by smooth muscle and endothelial cells over time to produce a membrane containing physiologic proteins that allows the extravasation under appropriate conditions.
 14. A method of assaying an agent for its ability to cause drug induced vascular injury, the steps comprising: a) providing an in vitro blood vessel model including two channels separated by a porous membrane coated on one side by an endothelial cell layer and coated on the other side by a smooth muscle cell layer, wherein the model is susceptible to the extravasation of red blood cells across the porous membrane due to drug induced vascular injury; b) passing the agent through the channel adjacent the endothelial cells; and c) analyzing the channel adjacent the smooth muscle cells for red bloods cell extravasation, which indicates that the agent causes drug induced vascular injury.
 15. The method of claim 14 wherein the porous membrane has pore sizes ranging from approximately 0.1 to 30 μm and a porosity between approximately 5 and 50 percent.
 16. The method of claim 14 wherein the porous membrane is a track-etched polycarbonate membrane.
 17. The method of claim 14 wherein step a) further includes passing medium through the two channels at arterial flow rates. 